The critical role of Y chromosome in sex determination and male fertility is one of the basic paradigms in reproductive biology. The most frequent cause of etiopathogenetic male infertility revealed by molecular genetic techniques is a deletion of the Y chromosome. The absence of genetic recombination of the Y chromosome makes it impossible to perform traditional positional mapping and cloning of the Y genes responsible for male germ cell progression. Deletion analysis of the human and mouse Y chromosome suggested several critical regions involved in male fertility. In mice most of the genes responsible for spermatogonial maintenance and germ cell differentiation are located on the short arm of the Y chromosome. One of such genes (Eif2s3y) was identified by transgenic gene rescue approach;however more recent data indicate that the additional genes within this deletion interval participate in spermatogenic progression. Further transgenic rescue experiments however become highly complicated as a production of mice with multiple transgenes on Y chromosome deletion background is required. Despite numerous attempts Y chromosome gene targeting using conventional ES cell technology did not result in live mouse mutant. We suggest here to target Y chromosome gene expression by production of mice with short hairpin RNA transgene. The activation of shRNA transgene expression and subsequent RNA interference in the proposed experiments is controlled by cre-recombinase, thus allowing transgene germ line transmission and specific gene targeting at different spermatogenic stages. This approach also allows targeting both single and multiple copy Y chromosomal genes, with a strict control of RNAi specificity. Our main hypothesis is that the genes located on the Y chromosome contribute to male germ cell maintenance and differentiation. The overall objective of current project is to characterize the spermatogenic functions of the Y chromosome genes through production and analysis of reproductive characteristics of shRNA transgenic males.To address this goal we designed two specific aims: 1. To produce conditional shRNA transgenic mice targeting eight genes from the short arm of the Y chromosome. 2. We will analyze the effect of Y chromosome gene suppression on spermatogenesis in males with shRNA transgene activated at different stages of male germ cell differentiation. Information obtained from these studies will serve as a basis for future studies in accurate genetic diagnostics of male infertility, the knowledge-based etiological therapy of symptoms associated with Y chromosome deletion, as well as contraceptive research. PUBLIC HEALTH RELEVANCE: The critical role of Y chromosome in sex determination and male fertility is one of the basic paradigms in reproductive biology. We propose to study the spermatogenesis and germ cell renewal in novel transgenic mice with targeted down-regulation of the Y chromosomal genes.